Studies in the field of muscle Biophysics, Physiology, Nano-Scale Physics and Mechanics have been performed traditionally with preparations ranging from the whole muscle to isolated muscle cells. While these preparations allow measurements of force and muscle mechanics with high reproducibility, they do not allow the investigation of sub-cellular units—myofibrils, sarcomeres, molecules, etc. Recently, studies with myofibrils and sub-cellular units of muscles have emerged—but giving the complexity of the experiments involving these structures, only a few laboratories around the world have this capability.
Furthermore, the systems used so far with myofibrils have several limitations, including a low signal-to-noise ratio, which limits detection of small differences in force observed in different conditions, a very low time resolution which limits the detection of molecular and kinetics events that may happen at the microsecond scale, and the incapacity of measuring force and imaging the myofibrils simultaneously with high spatial and temporal resolution.
An ideal system should be able to provide measurements of minute force measurements in the attonewton (aN) to nanonewton (nN) range in myofibrils with microsecond scale temporal resolution, and high signal-to-noise ratio. From a biological perspective, measurements of molecular kinetics with unprecedented precision are highly desirable. For example, measurements of the kinetics of myofibrils and myofilaments composed of muscle molecules with the time resolution of microseconds should open new opportunities in the fields of physiology and biophysics.
At present the dominant microscope for such measurements and analysis is the Atomic Force Microscope (AFM) because the most evident advantage that atomic force microscopy boasts over all other microscopic and nanoscopic imaging methods is its unique ability to probe forces. A cantilever acts as the force transducer; its interaction with a sample causes a deflection which can be measured and calibrated into a force. The versatility of AFM is highlighted by the fact that the same instrument can be used for probing the piconewton forces of a single covalent bond for example through to studying the forces exerted by cells in the micronewton range.
However, all commercial AFMs are optimized for measuring forces which are perpendicular to the sample surface, which becomes a limitation when the forces of interest are parallel to the sample surface. This has led many researchers to design home-built systems to overcome this problem by the use of the pendulum geometry, wherein the cantilever is positioned with its long axis perpendicular to the sample surface, such that forces in-plane can be measured with high sensitivity. The pendulum geometry prevents the snap-to-contact problem that afflicts soft cantilevers in a regular AFM. Very soft cantilevers enable attonewton force sensitivity which is necessary, for example, in the detection of single spins in magnetic resonance force microscopy (MRFM). At the microscopic length scale, the studies of cellular or subcellular forces which are parallel to the imaging plane of an optical microscope place system performance requirements for high sensitivity force measurements at sampling frequencies beyond available video rates, requirements that can be met by optical measurement techniques.
However, the difficulty in implementing the pendulum geometry lies in the constraint imposed by the focused incoming light or the diverging outgoing light which easily interferes with the sample surface. Additionally in order to obtain the measurements of biological tissue samples in vitro the optical measurement system must satisfy complex physical constraints to provide access to the vertical cantilever and its holder with consideration of solution ingress/egress, temperature control, and pipette for stimulation of the biological sample. Accordingly it would be beneficial to provide an optical measurement system which overcomes these complex physical constraints. According to an embodiment of the invention the geometrical restriction is addressed by the exploitation of an optical periscope.
Beneficially such a system may be employed for many other biological and physical applications and far-reaching potential for studies using measurements of light displacement towards cantilevers in constrained spaces. It can be used to expand the capabilities of atomic force microscopy in all of its current fields of study, such as biophysics, biology, surface science, and the emerging field of magnetic resonance force microscopy (MRFM). Consequently it would be beneficial to provide a system and a method that resolves the aforementioned limitations which will enable the desired measurements.